Studies On Preparation, Bioactivities And Application Of Phytosterols Ester

Phytosterols, including J3-sitosterol, stigmasterol, campesterol and brassicasterol mainly, were obtained commonly from by-product of refining vegetable oil. Phytosterols have been use as a novel functional food ingredient with plasma cholesterol lowering activity which works by inhibiting the absorption of cholesterol. Utilizations of free phytosterols in food were limited because of their insolubility in water or edible fats/oils. In order to improve solubility of free phytosterols in edible fats/oils, preparation technology, analytic methods, bioactivities and applications of phytosterols ester were studied systematically in this dissertation. The main results are shown as follows:1. Study on analytic methods of plytosterols and plytosterols esterGas-chromatography(GC) method for the quantitative determination of phytosterols was developed. Gas-chromatography separate conditions were selected as follows: HP-5 capillary gas-chromatography column; flame ionization detector(FID); injector 300℃; oven temperature 285℃; squalane as internal standard. The method has the advantages of exact and quick.The preparation and purification of mixed plytosterols standard by a recrystallization method were studied, best solvents and operate conditions were found .The experimental results showed that the total content of plytosterols was higher than 99% after the third recrystallization operation .The applications of mixed plytosterols standard in quantitative analysis by gas chromatography were researched , and these methods were convenient, short-cut, cheap and practical.The determination method of phytosterols ester was established. Methanol sodium as catalyzer, phytosterols ester sample were interesterificated with methanol. The compose and relatively content of fatty acid methyl ester were determinated by GC, accordingly, species and content of fatty acid could been make sure. Phytosterols ester sample were saponificated in KOH-alcohol solution, reaction temperature 80℃, reaction time 2h. Phytosterols ester were translated into free phytosterols. Then, through determinated the content of free phytosterols by GC, make a conversion to the content ofcorresponding phytosterols ester respectively.UV spectrum, IR spectrum and GC-MS were used to determine the nature the phytosterols and phytosterols ester. GC-MS crack law of phytosterols and phytosterols esters were summarized.2. Study on preparation of plytosterols esterUsing pyridine as catalyst, phytosterols acetate (PSA) were synthesized by direct esterification of phytosterols and acetic anhydride.The effect of factors on the yield was studied.The optimal synthetic conditions obtained were as follows: molar ratio of phytosterols to acetic anhydride to pyridine was above 1:9:5; Reaction temperature was 85°Cand reaction time was 2h. The recrystallization method phytosterols acetate were researched. Results of GC analysis of the product show that the purity of product was above 98%.Phytosterols oletate (PSO) were synthesized by direct esterification of phytosterols and oleic acid.The optimal synthetic conditions obtained were as follows: using sodium bisulfate as catalyst(2.0% molar of phytosterols);molar ratio of oleicacid to phytosterols was 1.3, Reaction temperature was 135 °C and reaction time was 8h. the yield of esterify reached 84.3% , the purity of product was above 90%.Phytosterols stearic (PSS) were synthesized by direct esterification of phytosterols and stearic acid .Through factorial design, the optimal synthetic conditions were obtained as follows: molar ratio of phytosterols to stearic acid was above 1:1.2; reaction temperature was 135°C and reaction time was 7h . The yield of esterification was 87.8%. Recrystallization method of phytosterols stearic were researched , the purity of product was above 95%.3. Study on serum lipid-lowering bioactivities of plytosterols esterStudy on the preventive and therapeutic effects of three phytosterol esters (PSA> PSO -. PSS) on diet-induced hyperlipidemia in mice and their structure-activity relationships. On preventive experiments, fourty healthy, male Kunming mice were randomly divided into five groups on body weight, eight mice per group. The control groups were fed with normal diet, while hyperlipidemic group were fed with the high-cholesterol diet. The rest three groups were given the high-cholesterol diets, at the same time, they were respectly gavaged with three phytosterol esters at the level of lOOmg/kg body weight. The effects of phytosterol esters on lipid metabolism in mice was studied. The experiment lasted twenty-eight days. Results showed that compared with the hyperlipidemic group, phytosterol esters could significantly lower the cholesterol levels of mice, incloud the serum TC, LDL-C and arteriosclerosis index (AI) . They also could lower the liver weight , TC and TG. PSA and PSO had ideal preventive effects ondiet-induced hyperlipidemia in mice and better than PSS.On therapeutic experiment, Kunming mice were fed with the high-cholesterol diet to form the hyperlipidemic model after 21 days.Then they were fed with normal diet, at the same time, the hyperlipidemic model group were gavaged with physiology brine, the medication group were gavaged with xuezhikang(20mg/kg-d), the rest three groups were gavaged with three phytosterol esters (100mg/kg-d) respectively. The effects of phytosterol esters on lipid metabolism in mice was studied. Results showed that compared with the hyperlipidemic model group, phytosterol esters could significantly reduce the serum TC, LDL-C and AI, lower the liver weight , TC and TG There are different active effects among three phytosterol esters, PSA and PSO had ideal treatment effects on hyperlipidemia in mice and better than PSS. Phytosterol esters have the function of regulating blood fat and ideal treatment effects on hyperlipidemia in mice . 4. Study on effect of inhibiting tumor and function of immune enhancement of plytosterols esterInvestigated the anti-tumor effect of Phytosterol Acetate(PSA) and Phytosterols Oletate(PSO). PSA (10,50mg/kg-d) and PSO (10,50mg/kg-d) were given respectively to mice bearing transplanted tumor Siso by intraperitoneal injection for 9 days.The inhibiting rate of tumor growth, thymus index, spleen index, activity of catalase were detected. Results showed that both PSA and PSO group could inhibit the grouth of Siso cell efficiently and increase the activity of catalase in erythrocyte of Sigo-bearing mice. Compared with cyclophosphamid, they did not decrease the immune viscera weight and interference the growth of Siso-bearing mice. The results in vitro showed that both PSA and PSO could inhibit the grouth of Siso cell in mice, exhibiting dosage-depended. Effect of inhibiting tumor of PSO were better than PSA.Evaluated the effects of phytosterol oletate (PSO) on the immune function of S180-bearing mice. Fifty mice bearing transplanted tumor Siso were used as animal model, The effects of PSO on the immune function were observed by intraperitoneal injecting different doses of PSO-1 (10 mg/kg-d), PSO-2 (50 mg/kg-d) and Cyclophosphamide (20 mg/kg-d) to mice respectively. Compared with the S18o-bearing mice model group, PSO could enhance significantly the phagocytosing ratio and the phagocytosing index (p<0.05), the effects of Si8o were better than Cyclophosphamide; PSO also could improve evidently delayed-type hypersensitivity (p<0.05), but worse than Cyclophosphamide; PSO could increased formation of antibody in splenic cells of Siso-bearing mice, but there is not remarkable different (p>0.05); PSO had little influence on the contents of serum hemolysin (p>0.05). The results showed that PSO could improved immunologic function of mice.5. Study on application of plytosterols esterStudy on the soluble capacity of phytosterols ester and phytosterols. There are significant different in the soluble capacity among three phytosterols esters . Solubility(g/100mL,20°C)of phytosterols in soy salad oil was 1.55, PSO was 31.8, PSA was 8.07, PSS was 1.82; The soluble capacity of solubility was: PSO PSA > PSS > phytosterols.The functional mayonnaise could be make up by adding PSO(2.5%) and natural tocopherol(0.5%) in the ordinary mayonnaise food. The optimum ingredients of the functional mayonnaise product were as follows: special soy salad oil(include PSO 3.5%, Ve 0.7%)74 shares, yolk 14 shares and vinegar 9 shares.